Breast cancer is the most common cancer and the second leading cause of cancer death in women. A better understanding of breast cancer etiology would help us to better prevent and treat this disease. The LBK1-AMPK pathway is a central regulator of energy metabolism, and misregulation of this pathway has been implicated in cancers, including breast cancer. Indirect AMPK activators such as metformin have shown beneficial effect in breast cancer prevention and treatment. However, other than LKB1 mutations, how this pathway might be misregulated in breast cancer remains unclear. In this application, based on our extensive Preliminary Data, we propose to characterize several factors based on their newly identified roles in AMPK regulation and in turn, on breast cancer and treatment response. The first factor is called tumor suppressor protein 52 (TPD52) which we found to negatively regulate AMPK. TPD52 is known to overexpress in HER2+ breast cancer and, together with HER2, to promote tumor growth. The AMPK pathway is also known to be involved in anti-HER2 response. Furthermore, we found that AMPK regulates USP10. USP10 can enhance the stability of two important tumor suppressors, p53 and SIRT6, so our finding has established a new downstream pathway to AMPK. It is reported that SIRT6 can also activate AMPK. Therefore, we have identified a new axis of AMPK regulation with TPD52 negatively regulating AMPK with the AMPK downstream factors, UPS10/SIRT6 forming a positive feedback loop, further activating AMPK, all of which would be important in energy metabolism and response to metabolic stress induced by metformin. Metformin response is heterogeneous; therefore our findings could shed light on the underlying mechanisms. Our preliminary data indicated that cells with TPD52 overexpression were more sensitive to metformin as well as combined metformin+HER2 inhibitors. This is consistent with previous studies indicating that metformin can inhibit cell proliferation and increase patient survival in HER2+ breast cancer. Based on these preliminary findings, we hypothesize that the AMPK-USP10 axis contributes to the metabolic function of AMPK; TPD52, by negatively regulating AMPK, can promote misregulation of energy metabolism and contribute to breast cancer development. Drugs like metformin would be more effective in patients with TPD52 overexpression and might be used in combination with HER2 inhibitors in HER2+ cancers. Therefore, in this application, we propose to determine how TPD52 regulates the AMPK pathway and how AMPK might regulate SIRT6 and p53 through the regulation of USP10 as well as the impact of this regulation on response to biguanides and anti-HER2 therapy using cell lines, breast tumor samples, and breast cancer patient derived xenografts. In summary, these studies would help us to understand how this new axis TPD52-AMPK-USP10-SIRT6/p53 contributes to tumorigenesis and treatment response.